Antimicrobial compounds

ABSTRACT

Disclosed herein are compounds for use as antimicrobial agents, having a structure of formula (I):

FIELD OF THE INVENTION

The present invention relates to novel aryl compounds. More particularlythe present invention relates to novel aryl compounds and their use asantimicrobials to treat bacterial infections or diseases.

BACKGROUND ART

Compounds with antimicrobial properties have attracted great interest inrecent times as a result of an increase in the prevalence of infectionscaused by Gram-positive bacteria, resulting in serious or fataldiseases. Furthermore, the regular use of broad spectrum antibioticformulas has led to the increased occurrence of bacterial strainsresistant to some antimicrobial formulations.

Novel antimicrobial compounds have the potential to be highly effectiveagainst these types of treatment-resistant bacteria. The pathogens,having not previously been exposed to the antimicrobial formulation, mayhave little to no resistance to the treatment.

DISCLOSURE OF THE INVENTION General

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variation and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in the specification, individually or collectively andany and all combinations or any two or more of the steps or features.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally equivalent products, compositions andmethods are clearly within the scope of the invention as describedherein.

The entire disclosures of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference, whichmeans that it should be read and considered by the reader as part ofthis text. That the document, reference, patent application or patentcited in this text is not repeated in this text is merely for reasons ofconciseness. No admission is made that any of the references constituteprior art or are part of the common general knowledge of those workingin the field to which this invention relates.

Throughout this specification, unless the context requires otherwise,the term antimicrobial is understood to include compounds withantibacterial properties.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers.

Other definitions for selected terms used herein may be found within thedetailed description of the invention and apply throughout. Unlessotherwise defined, all other scientific and technical terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the invention belongs.

Use of compounds

In one form, the present invention provides for the use of a compound ofFormula A, or a pharmaceutically acceptable salt thereof in themanufacture of a medicament for the therapeutic treatment of bacterialinfection or disease in a subject in need thereof.

Where;

-   -   W is a linking unit and Z is a peripheral unit. V, linking unit        W and peripheral unit Z are all linked by co-valent bonds;    -   n=1, 2, 3, or 4.    -   V=a six membered aromatic ring.

The, or each W may be independently selected from:

-   -   i. C₂₋₄ alkyl groups;    -   ii. C₂₋₄ substituted alkyl groups;    -   iii. C₂ E-alkene, with V and Z in the 1 and 2 positions;    -   iv. C₂ Z-alkene, with V and Z in the 1 and 2 positions;    -   v. C₂-alkyne;

The, or each Z may be selected from any one or more of:

R, may be independently selected from any one or more of i-xxxvii, withat least one R group being independently selected from any one of ii-xxxvii:

-   -   i. —H,    -   ii. —Halo,    -   iii. C₁₋₈ alkyl,    -   iv. C₁₋₈ heteroalkyl,        -   a) Carboxylic acids and related derivatives independently            selected from:        -   b) Carboxylic acid,

-   -   -   c) Thiocarboxylic acid,

-   -   -   d) Esters,

-   -   -   e) Thioester,

-   -   -   f) Dithioester,

-   -   v. Amide derivatives of carboxylic acids independently selected        from:        -   a) Amide,

-   -   -   b) Thioamide,

-   -   vi. Aldehydes, ketones and their derivatives independently        selected from:        -   a) Aldehyde,

-   -   -   b) Thial,

-   -   -   c) Ketones,

-   -   -   d) Thioketones,

-   -   -   e) Acetals,

-   -   -   f) Dithioacetals,

-   -   vii. Amines, alkyl amines and their derivatives independently        selected from        -   a) Amines,

-   -   -   b) Amides,

-   -   -   c) Thioamide,

-   -   -   d) Ammonium salts,

-   -   -   e) Alkyl amines,

where n=1-3

-   -   -   f) Alkyl amides,

where n=1-3

-   -   -   g) Alkyl thioamides,

where n=1-3

-   -   -   h) Alkyl ammonium salts,

where n=1-3,

-   -   -   i) Imines,

-   -   -   j) Guanidines,

-   -   -   k) Amidine,

-   -   viii. Nitrile (cyano), —C≡N,    -   ix. Isonitrile, —N⁺═C⁻,    -   x. Cyanate, —O—C≡N,    -   xi. Isocyanate, —N═C═O,    -   xii. Thiocyanate, —S—C≡N,    -   xiii. Isothiocyanate, —N═C═S,    -   xiv. Azo, —N═NH ,    -   xv. Nitro,

-   -   xvi. Nitrite, —O—N═O,    -   xvii. Nitriso, —N═O,    -   xviii. N-terminal peptide sequences,

where x=1-3 and RP_(pep) is any group resulting in the formation of anamino acid.

-   -   xix. C-terminal peptide sequences,

where x=1-3 and RP^(pep) is any group resulting in the formation of anamino acid.

-   -   xx. Phosphorus based substituents, where the phosphorus atom is        in either the 3+ or 5+ oxidation state, independently selected        from:        -   a) Phosphines,

-   -   -   b) Phosphine oxides,

-   -   -   c) Phosphites,

-   -   -   d) Phosphates,

-   -   -   e) Phosphinites,

-   -   -   f) Phosphinates,

-   -   -   g) Phosphinites,

-   -   -   h) Phosphonates,

-   -   xxi. sulfur based substituents,        -   a) Sulfate,

-   -   -   b) Sulfone,

-   -   -   c) Sulfoxide,

-   -   -   d) Sulfinic acids,

-   -   -   e) Sulfimines,

-   -   -   f)Sulfonamides,

-   -   xxii. Boron based substituents,        -   a) Boronic acids,

-   -   -   b) Boronic esters,

-   -   xxiii. Semicarbazones,

-   -   xxiv. Thiosemicarbazones,

-   -   xxv. Cyanimide,

-   -   xxvi. Hydrazone,

-   -   xxvii. Oxime,

-   -   xxviii. Nitroamine,

-   -   xxix. Nitronate,

-   -   xxx. Nitrone,

-   -   xxxi. Carbonates,

-   -   xxxii. Carbamates,

-   -   xxxiii. Dithiocarbamates,

-   -   xxxiv. A 5- or 6- membered saturated heterocyclic ring        containing 1 to 3 heteroatoms chosen independently from        nitrogen, sulfur and oxygen.    -   xxxv. A 5- or 6- membered saturated heteroaromatic ring        containing 1 to 5 heteroatoms chosen independently from        nitrogen, sulfur and oxygen.    -   xxxvi. A 5- membered unsaturated heterocyclic ring containing,        one or two double bonds and containing 1 to 5 heteroatoms chosen        independently from nitrogen, sulphur and oxygen.    -   xxxvii. A 6 membered ring containing 1 to 3 double bonds and        containing1 to 5 heteroatoms chosen independently from nitrogen,        sulfur and oxygen dependant on ring size.

The present invention further provides for the use of a compound ofFormula B:

or a pharmaceutically acceptable salt thereof in the manufacture of amedicament for the therapeutic treatment of bacterial infection ordisease in a subject in need thereof, wherein:

-   -   n′=2, 3 or 4    -   R′ comprises any one of C₂-alkyl, C₂-alkene or C₂-alkyne; and    -   R″ is independently selected from NH₂, CH₃, OH, —COCH₃,        —OC(CH₃)₃, OCH₃, OCH₂CH₃, OCH(CH₃)₂, —CONH₂, COOH, COOCH₃, or        COOCH₂CH₃

In a preferred form, the present invention provides for the use of acompound of Formula B:

or a pharmaceutically acceptable salt thereof in the manufacture of amedicament for the therapeutic treatment of bacterial infection ordisease in a subject in need thereof wherein:

-   -   n′=2, 3 or 4    -   R′=C₂-alkyl or C₂-alkene    -   R″ is independently selected from COOCH₃, COOCH₂CH₃ or COOH

In a further preferred form, the invention provides for the use of acompound of Formula C:

or a pharmaceutically acceptable salt thereof in the manufacture of amedicament for the therapeutic treatment of bacterial infection ordisease in a subject in need thereof.

The compound of Formula C is also known as NAL135B, 135B and TSB007 (seeFIG. 7).

In an alternative aspect, the compound provided for the use of acompound selected from the following list of analogues of formula C:T5B049, T5B063, T5B037, TSB041, TSB019, T5B023, T5B025, T5B065, TCT003,TSB001, TSB009, T5B053, TAB001, TCB001, TCB003 (see FIGS. 8 to 12 forstructure information). More preferably, the compound is T5B065 orTCB001.

The use of a compound of any one of Formulas A, B or C, or analoguesthereof, or pharmaceutically acceptable salts thereof in the manufactureof a medicament for the therapeutic treatment of bacterial infection ordisease in a subject in need thereof, wherein the bacterial infection ordisease results from Gram-positive bacteria.

Method of Treatment

The compounds of the present invention have antimicrobial activity, andthus are useful for treatment of bacterial infections. Thus, the presentinvention also provides a method of treating a bacterial infection in asubject comprising the step of administering to the subject an effectiveamount of a compound of Formula A and variants described herein.

The present invention further provides a method of treating a bacterialinfection in a subject comprising the step of administering to thesubject an effective amount of a compound of Formula B:

Wherein:

-   n′=2, 3 or 4-   R′ comprises any one of C₂-alkyl, C₂-alkene or C₂-alkyne; and

R″ is independently selected from NH₂, CH₃, OH, —COCH₃, —OC(CH₃)₃, OCH₃,OCH₂CH₃, OCH(CH₃)₂, —CONH₂, COOH, COOCH₃, or COOCH₂CH₃.

In a preferred form the present invention provides a method of treatinga bacterial infection in a subject comprising the step of administeringto the subject an effective amount of a compound of Formula B wherein:

-   n′=2, 3 or 4-   R′=c₂-alkyl o C₂-alkene-   R″ is independently selected from COOCH₃, COOCH₂CH₃ or COOH

In a further preferred form, the present invention provides a method oftreating a bacterial infection in a subject comprising the step ofadministering to the subject an effective amount of a compound ofFormula C:

In an alternative aspect, the present invention provides a method oftreating a bacterial infection in a subject comprising the step ofadministering to the subject an effective amount of a compound selectedfrom the following list of analogues of formula C: T5B049, T5B063,T5B037, TSB041, TSB019, T5B023, T5B025, T5B065, TCT003, TSB001, TSB009,T5B053, TAB001, TCB001, TCB003 (see FIGS. 8 to 12 for structureinformation). More preferably, the compound is T5B065 or TCB001.

The compounds of the present invention are understood to be effectiveagainst both Gram positive bacteria and Gram negative bacteria.

Examples of Gram-negative bacteria which the compounds of the presentinvention may preferably be used against include: Aeromonas hydrophila,Citrobacter freundii, Escherichia coli, Klebsiella edwardsii, Proteusmirabilis, Salmonella enterica subsp. enterica serovar Typhimurium,Moraxella catarrhalis, Shigella flexneri, Stenotrophomonas maltophilia,Vibrio cholerae (non-toxigenic) and Yersinia enterocolitica.

The compounds of the present invention are understood to be mosteffective against Gram-positive bacteria. Examples of Gram-positivebacteria which the compounds of the present invention may preferably beused against include: Bacillus cereus, Bacillus subtilise, Clostridiumdifficile, Enterococcus faecalis, Enterococcus faecium, Listeriamonocytogenes, Micrococcus luteus, Staphylococcus aureus, Staphylococcusaureus (methicillin resistant), Staphylococcus epidermidis,Staphylococcus xylosus, Streptococcus pneumonia, and Streptococcuspyogenes.

The compounds of the present invention are understood to be particularlyeffective against methicillin-resistant Staphylococcus aureus (MRSA) andClostridium difficile.

Compositions

The compounds of the present invention may be formulated intocompositions for administration.

Thus, the present invention also provides a composition comprising atherapeutically-effective amount of a compound of Formula A and variantsdescribed herein, and a pharmaceutically acceptable carrier or diluent.

The present invention also provides a composition comprising atherapeutically-effective amount of a compound having a Formula B and apharmaceutically acceptable carrier or diluents:

-   n=2, 3 or 4-   R′ comprises any one of C₂-alkyl, C₂-alkene or C₂-alkyne; and-   R″ is independently selected from NH₂, CH₃, OH, -COCH₃, —OC(CH₃)₃,    OCH₃, OCH₂CH₃, OCH(CH₃)₂, —CONH₂, COOH, COOCH₃, or COOCH₂CH₃.

In a preferred form, the present invention provides a compositioncomprising a therapeutically-effective amount of a compound having aFormula B and a pharmaceutically acceptable carrier or diluents

wherein:

-   n=2, 3 or 4-   R′ =c₂-alkyl o C₂-alkene-   R″ is independently selected from COOCH₃, COOCH₂CH₃ or COOH.

The present invention also provides a composition comprising atherapeutically-effective amount of a compound having a Formula C and apharmaceutically acceptable carrier or diluent.

In an alternative aspect of the present invention, there is provided acomposition comprising a therapeutically-effective amount of a compoundselected from the following list of analogues of compound C: T5B049,T5B063, T5B037, TSB041, TSB019, T5B023, T5B025, T5B065, TCT003, TSB001,TSB009, T5B053, TAB001, TCB001, TCB003 (see FIGS. 8 to 12 for structureinformation) and a pharmaceutically acceptable carrier or diluent. Morepreferably, the compound is T5B065 or TCB001.

With respect to use, methods of treatment and compositions comprisingthe compounds having a Formula A, B or C, or analogues thereof andvariants described herein:—

V is preferably, a phenyl or a pyridal ring.

Where V is a phenyl ring and:

-   -   i. n=3, the W—Z group may be substituted at positions 1, 2, and        3; or 1, 2, and 4; or 1, 3 and 5, with A¹, A² and A³ occupying        remaining positions on the ring:

-   -   ii. n=4, W—Z may be substituted at positions 1, 2, 3 and 4; 1,        2, 3 and 5; 1, 2, 4 and 5 with A¹ and A² occupying remaining        positions on the ring:

-   -   iii. n=5, W—Z may be substituted at positions 1, 2, 3, 4 and 5        with A¹ occupying the remaining position on the ring:

-   -   iv. n=6, W—Z is substituted into each of positions 1, 2, 3, 4, 5        and 6.

Where V is a pyridal ring, and:

-   -   i. n=3, W—Z may be substituted into positions 2, 3, and 4; or 2,        3, and 5; or 2, 3, and 6; or 2, 5, and 6; or 2, 4 and 6; or 3,        4, and 5, with A¹ and A² occupying the remaining positions on        the pyridal ring, and where the pyridal nitrogen (N) occupies        position 1.

-   -   ii. n=4, W—Z may be substituted into positions, 2, 3, 4, and 5;        or 2, 3, 5, and 6, with A¹ occupying the remaining position on        the pyridal ring, and where the pyridal nitrogen (N) occupies        position 1.

-   -   iii. n=5, W-Z may be substituted into positions, 2, 3, 4, 5 and        6; and where the pyridal nitrogen (N) occupies position 1.

Where the, or each W is in the form of a C₂ E-alkene, with V and Z inpositions 1 and 2, positions 3 and 4 are each preferably independentlysubstituted with a hydrogen or C₁-₄ alkyl.

Where the, or each W is in the form of a C₂ Z-alkene, with V and Z inpositions 1 and 2, positions 3 and 4 are each preferably independentlysubstituted with a hydrogen or C₁-₄ alkyl.

Where R_(n) is a carboxylic acid, the carboxylic acid is preferably inthe form of COOR^(a).

More preferably, R_(a) is any one of hydrogen, alkyl (C₁₋₄).

Where R_(n) is an amide derivative of a carboxylic acid, R^(a) and R^(b)are independently selected from hydrogen or alkyl (C₁₋₄).

Where R_(n) is an aldehyde, ketone, or a derivative thereof, R^(a) andR^(b) are independently selected from hydrogen or alkyl (C₁₋₄).

Where R_(n) is an amine, alkyl amine or a derivative thereof, R^(a),R^(b), and R^(c) are independently selected from hydrogen or alkyl(C₁₋₄).

Where R_(n) is a saturated 5 or 6 membered heterocyclic ring, theheterocycle is preferably any one of piperidinyl, morpholinyl,thiomorpholinyl, thiazolidinyl, isothiazolidinyl, oxazolidinyl,isooxazolidinyl, pyrrolidinyl, imidazolidinyl, piperazinyl,tetrahydrofuranyl, dioxaanyl, dithanyl, and pyrazolidinyl.

The saturated heterocyclic ring may be unsubstituted. Alternatively, thesaturated heterocyclic ring may be substituted with 1 to 3 substituentsindependently selected from:

-   -   1. Halogen, independently selected from Br, I, Cl, F    -   2. Alkyl, independently selected from methyl, ethyl, propyl or        butyl

Where R_(n) is a 5 or 6 membered heteroaromatic ring, the heteroaromaticis preferably any one of thienyl, pyridyl, imidazolyl, pyrrolyl,pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isooxazolyl, pyrazinyl,pyrimidinyl, triazolyl, tetrazolyl, furanyl, triazinyl, and pyridazinyl.

The heteroaromatic ring may be unsubstituted. Alternatively, theheteroaromatic ring may be substituted with 1 to 5 substituentsindependently selected from:

-   -   1. Halogen, independently selected from Br, I, Cl, F    -   2. Alkyl, independently selected from methyl, ethyl, propyl or        butyl

Where R_(n) is an unsaturated 5 or 6 membered heterocyclic ring, theheterocycle is preferably any one of oxolanyl, imidazolinyl,pyrazolinyl, thiazolidinyl, oxazolininyl, dithiolanyl, and dioxolanyl.

More preferably, the unsaturated heterocyclic ring is not an aromatic.

The unsaturated heterocyclic ring may be unsubstituted. Alternatively,the unsaturated heterocyclic ring may be substituted with 1 to 5substitutents independently selected from:

-   -   1. Halogen, independently selected from Br, I, Cl, F    -   2. Alkyl, independently selected from methyl, ethyl, propyl or        butyl.

R′ is preferably in the form of any one of C₂-alkyl o C₂-alkene, and R″is independently selected from COOCH₃, COOCH₂CH₃ or COOH.

Preferably, where n′=2, R′—C₆H₄—R″, is substituted at positions 1 and 3.

Preferably, where n′=3, R′—C₆H₄—R″, is substituted at positions 1, 3 and5; or 1, 2 and 4.

Preferably, where n′=3, R′—C₆H₄—R″, is substituted at positions 1, 2, 4and 5.

RP^(pep) preferably has a peptide comprised of one or more α-aminoacids, independently selected from alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine or valine.

The present invention provides use of a compound of Formulas A, B or C,or analogues thereof, or pharmaceutically acceptable salts thereof inthe manufacture of a medicament for the therapeutic treatment ofbacterial infection or disease in a subject in need thereof, wherein thebacterial infection or disease results from Gram-positive bacteria.

The precise composition of the present invention will vary according toa wide range of commercial and scientific criteria. Methods for thepreparation of pharmaceutical compositions comprising one or more activeingredients are generally known in the art. Such compositions willgenerally be formulated for the mode of delivery that is to be used andwill usually include one or more pharmaceutically acceptable carriers.

Generally, examples of suitable carriers, excipient and diluentsinclude, without limitation, water, saline, ethanol, dextrose, glycerol,lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphates, alginate, tragacanth, gelatine, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, watersyrup, methyl cellulose, methyl and propylhydroxybenzoates, talcmagnesium stearate and mineral oil or combinations thereof. Theformulations can additionally include lubricating agents, pH bufferingagents, wetting agents, emulsifying and suspending agents, preservingagents, sweetening agents or flavouring agents.

(a) Topicals

The pharmaceutical composition may be adapted for topical application.In this regard, various topical delivery systems may be appropriate foradministering the compositions of the present invention depending up onthe preferred treatment regimen. Topical formulations may be produced bydissolving or combining the compound of the present invention in anaqueous or nonaqueous carrier. In general, any liquid, cream, or gel orsimilar substance that does not appreciably react with the compound orany other of the active ingredients that may be introduced into thecomposition and which is non-irritating is suitable. Appropriatenon-sprayable viscous, semi-solid or solid forms can also be employedthat include a carrier compatible with topical application and havedynamic viscosity preferably greater than water.

Suitable formulations are well known to those skilled in the art andinclude, but are not limited to, solutions, suspensions, emulsions,creams, gels, ointments, powders, liniments, salves, aerosols,transdermal patches, etc, which are, if desired, sterilised or mixedwith auxiliary agents, e.g. preservatives, stabilisers, emulsifiers,wetting agents, fragrances, colouring agents, odour controllers,thickeners such as natural gums, etc. Particularly preferred topicalformulations include ointments, creams or gels.

Ointments generally are prepared using either (1) an oleaginous base,i.e., one consisting of fixed oils or hydrocarbons, such as whitepetroleum or mineral oil, or (2) an absorbent base, i.e., one consistingof an anhydrous substance or substances which can absorb water, forexample anhydrous lanolin. Customarily, following formation of the base,whether oleaginous or absorbent, the active ingredient is added to anamount affording the desired concentration.

Creams are oil/water emulsions. They consist of an oil phase (internalphase), comprising typically fixed oils, hydrocarbons and the like,waxes, petroleum, mineral oil and the like and an aqueous phase(continuous phase), comprising water and any water-soluble substances,such as added salts. The two phases are stabilised by use of anemulsifying agent, for example, a surface active agent, such as sodiumlauryl sulfite; hydrophilic colloids, such as acacia colloidal clays,veegum and the like. Upon formation of the emulsion, the compound can beadded in an amount to achieve the desired concentration.

Gels comprise a base selected from an oleaginous base, water, or anemulsion-suspension base. To the base is added a gelling agent thatforms a matrix in the base, increasing its viscosity. Examples ofgelling agents are hydroxypropyl cellulose, acrylic acid polymers andthe like. Customarily, the compound is added to the formulation at thedesired concentration at a point preceding addition of the gellingagent.

The amount of compound incorporated into a topical formulation is notcritical; the concentration should be within a range sufficient topermit ready application of the formulation such that an effectiveamount of the compound is delivered.

(b) Oral Formulations

The pharmaceutical composition may be adapted for oral delivery. In thisregard, the compound can be administered as an oral preparation adaptedin such a manner that facilitates delivery of a therapeuticallyeffective concentration of the compound.

The effective dosages of the compound, when administered orally, musttake into consideration the diluent, preferably water. The compositionpreferably contains 0.05% to about 100% by weight active ingredient andmore preferably about 10% to about 80% by weight. When the compositionsare ingested, desirably they are taken on an empty stomach.

Contemplated for use herein are oral solid dosage forms includingtablets, capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate thepresent compositions. Liposomal encapsulation may be used and theliposomes may be derivatised with various polymers. In general, theformulation will include the compound and inert ingredients that allowfor protection against the stomach environment and release of thebiologically active material in the intestine.

The location of release may be the stomach, the small intestine (theduodenum, the jejunem, or the ileum), or the large intestine. Oneskilled in the art has available formulations that will not dissolve inthe stomach, yet will release the material in the duodenum or elsewherein the intestine. Preferably, the release will avoid the deleteriouseffects of the stomach environment, either by protection of thecomposition or by release of the compound beyond the stomachenvironment, such as in the intestine.

To ensure full gastric resistance, a coating impermeable to at least pH5.0 may be used. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings that are not intended for protectionagainst the stomach can also be used on tablets. This can include sugarcoatings, or coatings that make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatine) for delivery of drytherapeutic i.e. powder; for liquid forms, a soft gelatine shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, moulded tablets or tablet triturates,moist massing techniques can be used.

One may dilute or increase the volume of the composition with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, alpha-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the compound into asolid dosage form. Materials used as disintegrants include but are notlimited to starch including the commercial disintegrant based on starch,Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatine,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants is insoluble cationicexchange resins. Powdered gums may be used as disintegrants and asbinders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the composition together to form a hardtablet and include materials from natural products such as acacia,tragacanth, starch and gelatine. Others include methylcellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the compound.

An antifrictional agent may be included in the formulation to preventsticking during the formulation process. Lubricants may be used as alayer between the compound and the die wall and these can include butare not limited to: stearic acid including its magnesium and calciumsalts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oilsand waxes. Soluble lubricants may also be used such as sodium laurylsulfate, magnesium lauryl sulfate, polyethylene glycol of variousmolecular weights and Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the compositionduring formulation and to aid rearrangement during compression might beadded. The glidants may include starch, talc, pyrogenic silica andhydrated silicoaluminate.

To aid dissolution of the compound, a surfactant might be added as awetting agent. Surfactants may include anionic detergents such as sodiumlauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodiumsulfonate. Cationic detergents might be used and could includebenzalkonium chloride or benzethomium chloride. The list of potentialnonionic detergents that could be included in the formulation assurfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylenehydrogenated castor oil 10, 50 and 60, glycerol monostearate,polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methylcellulose and carboxymethyl cellulose. These surfactants could bepresent in the formulation either alone or as a mixture in differentratios.

Controlled release formulations may be desirable. The compounds can beincorporated into an inert matrix that permits release by eitherdiffusion or leaching mechanisms i.e., gums. Slowly degeneratingmatrices may also be incorporated into the formulation. Another form ofa controlled release formulation is by a method based on the Orostherapeutic system (Alza Corp.), i.e. the composition is enclosed in asemipermeable membrane which allows water to enter and push thecomposition out through a single small opening due to osmotic effects.Some enteric coatings also have a delayed release effect.

A mix of materials might be used to provide the optimum film coating.Film coating may be carried out in a pan coater or in a fluidised bed orby compression coating.

The compound can be included in the formulation as finemultiparticulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The compound could be prepared by compression.

(c) Injectable formulations

The compound can also be formulated for parenteral delivery.Pharmaceutical forms suitable for injectable use include: sterileaqueous solutions (where water-soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. Alternatively, the compounds of the inventionmay be encapsulated in liposomes and delivered in injectable solutionsto assist their transport across cell membrane. The solution may be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol and liquid polyethyleneglycol and the like), suitable mixtures thereof and vegetable oils.Proper fluidity may be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminium monostearate and gelatine.

Sterile injectable solutions may be prepared by incorporating the activecompounds in the required amount in an appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilisation. Generally, dispersions are prepared byincorporating the compound into a sterile vehicle that contains thebasic dispersion medium and the other ingredients. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingtechniques that yield a powder of the compound plus any additionaldesired ingredient from previously sterile-filtered solution thereof.

Thus, the present invention also provides an injectable, stable, sterilecomposition comprising a compound of Formula A, or a salt thereof, in aunit dosage form in a sealed container. The compound or salt may beprovided in lyophilised form capable of being reconstituted with asuitable pharmaceutically acceptable carrier to form a liquidcomposition suitable for injection thereof into a subject. The unitdosage form typically comprises from about 10 mg to about 10 grams ofthe compound or salt thereof. When the compound or salt is substantiallywater-insoluble, a sufficient amount of emulsifying agent which isphysiologically acceptable may be employed in sufficient quantity toemulsify the compound or salt in an aqueous carrier. One such usefulemulsifying agent is phosphatidyl choline.

(d) Aerosols

Pharmaceutical compositions are also provided which are suitable foradministration as an aerosol, by inhalation. These compositions comprisea solution or suspension of the desired compound or a salt thereof or aplurality of solid particles of the compound or salt. The desiredcomposition may be placed in a small chamber and nebulized. Nebulizationmay be accomplished by compressed air or by ultrasonic energy to form aplurality of liquid droplets or solid particles comprising the compoundsor salts.

The solid particles can be obtained by processing solid compound or asalt thereof, in any appropriate manner known in the art, such as bymicronization. Commercial nebulizers are also available to provideliquid droplets of any desired size.

The liquid droplets or solid particles should have a particle size inthe range of about 0.5 to about 5 microns, preferably from about 1 toabout 2 microns. Most preferably, the size of the solid particles ordroplets will be from about 1 to about 2 microns. Such particles ordroplets may be dispensed by commercially available nebulisers or byother means known to the skilled person.

When the pharmaceutical composition suitable for administration as anaerosol is in the form of a liquid, the composition will comprise awater-soluble form of the compound or a salt thereof, in a carrier thatcomprises water. A surfactant may be present which lowers the surfacetension of the composition sufficiently to result in the formation ofdroplets within the desired size range when subjected to nebulization.

In addition, the pharmaceutical composition may also include otheragents. For example, preservatives, co-solvents, surfactants, oils,humectants, emollients, chelating agents, dyestuffs, stabilizers orantioxidants may be employed. Water soluble preservatives that may beemployed include, but are not limited to, benzalkonium chloride,chlorobutanol, thimerosal, sodium bisulfate, phenylmercuric acetate,phenylmercuric nitrate, ethyl alcohol, methylparaben, polyvinyl alcohol,benzyl alcohol and phenylethyl alcohol. A surfactant may be Tween 80.Other suitable additives include lubricants and slip agents, such as,for example, magnesium stearate, stearic acid, talc and bentonites,substances which promote disintegration, such as starch or crosslinkedpolyvinylpyrrolidone, binders, such as, for example, starch, gelatin orlinear polyvinylpyrrolidone, and dry binders, such as microcrystallinecellulose.

Other vehicles that may be used include, but are not limited to,polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose, poloxamers,carboxymethyl cellulose, hydroxyethyl cellulose, purified water, etc.Tonicity adjustors may be included, for example, sodium chloride,potassium chloride, mannitol, glycerin, etc. Antioxidants include, butare not limited to, sodium metabisulfite, sodium thiosulfate,acetylcysteine, butylated hydroxyanisole, butylated hydroxytoluene, etc.The indications, effective doses, compositions, contraindications,vendors etc, of the compounds in the compositions are available or areknown to one skilled in the art. These agents may be present inindividual amounts of from about 0.001% to about 5% by weight andpreferably about 0.01% to about 2%.

Electrolytes such as, but not limited to, sodium chloride and potassiumchloride may also be included in the composition.

Further, the compositions may contain microbial preservatives. Usefulmicrobial preservatives include methylparaben, propylparaben, and benzylalcohol. The microbial preservative is typically employed when thecomposition is placed in a vial designed for multidose use.

Excipients which may be used are all the physiologically acceptablesolid inert substances, either inorganic or organic in nature. Inorganicsubstances are, for example, sodium chloride, carbonates, such ascalcium carbonate, bicarbonates, aluminium oxides, silicic acids,aluminas, precipitated or colloidal silicon dioxide and phosphates.Organic substances are, for example, sugars, cellulose, foodstuffs andfeedstuffs, such as milk powder, animal flours, cereal flours andshredded cereals and starches.

Finally, it will be appreciated that the compositions of the presentinvention may comprise a plurality of compounds as described herein.

Compounds

In one form, the invention provides a compound of Formula B:

Where:

-   N′=2, 3 or 4-   R′ comprises any one of C₂-alkyl, C₂-alkene or C₂-alkyne; and-   R″ is independently selected from NH₂, CH₃, OH, —COCH₃, —OC(CH₃)₃,    OCH₃, OCH₂CH₃, OCH(CH₃)₂, —CONH₂, COOH, COOCH₃, or COOCH₂CH₃.

In a preferred form, the invention provides a compound of Formula B:

wherein

R′ comprises any one of C₂-alkyl, C₂-alkene; and

R″ is independently selected from COOH, COOCH₃, or COOCH₂CH₃.

Preferably, where n′=2, R′-C₆H₄-R″, is substituted at positions 1 and 3.

Preferably, where n′=3, R′-C₆H₄-R″, is substituted at positions 1, 3 and5; or 1, 2 and 4.

Preferably, where n′=3, R′-C₆H₄-R″, is substituted at positions 1, 2, 4and 5.

In accordance with a further aspect of the present invention, there isprovided a compound of Formula C:

The compounds of the present invention may be provided in the form of apharmaceutically acceptable salt.

Pharmaceutically acceptable salts for the purposes of the presentinvention include non-toxic cation and anion salts. Examples include,but are not limited to sodium, potassium, aluminium, calcium, lithium,magnesium, zinc and from bases such as ammonium, ethylenediamine,N-methyl-glutamine, lysine, arginine, ornithine, choline, N,N′-dibenzylethlenediamine, diethylamine, piperazine,tris(hydroxymethyl)aminomethane, tetramethylammonium, acetate,lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate,maleate, bisulfate, mandelate, bitratrate, meyate, borate,methylbromide, bromide, methylnitrate, calcium edetate, methylsulfate,camsylate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate,N-methylglucamine, citrate, hydrochloride, oleate, edetate, oxalate,edisylate, pamoate (embonate), estolate, palmitate, esylate,pantothenate, fumarate, phosphate, diphosphate, glucepate,plygalacturonate, gluconate, salicylate, glutamate, stearate,glycollylarsanilate, sulfate, hexylresorcinate, subacetate, hydrabamine,succinate, hydrobromide, tannate, tartrate, hydroxynapthoate, teoclate,iodide, tosylate, isothionate, triethiodide, lactate, panoate andvalerate.

In an alternative aspect, the compound provided by the present inventionis preferably selected from the following list of analogues of compoundC: T5B049, T5B063, T5B037, TSB041, TSB019, T5B023, T5B025, T5B065,TCT003, TSB001, TSB009, T5B053, TAB001, TCB001, TCB003 (see FIGS. 8 to12 for structure information). More preferably, the compound is T5B065or TCB001.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 and 2 provide general pathways for synthesis of the targetcompounds through the Heck Cross coupling approach.

FIG. 3 is a disk diffusion analysis for TSB007 (135B) showing zoneinhibition at 48 hours for Staphylococcus aureus NCTC 10442 MRSA. Thedisk on the left contained 20 μl of 10 mg/ml 135B in DMSO and the diskon the right contained 20 μl of DMSO.

FIG. 4 is a disk diffusion analysis for TSB007 (135B), showing theinhibition at 48 hours for Streptococcus pneumoniae ATCC 49619. The diskon the left contained 20 μl of 10 mg/ml 135B in DMSO and the disk on theright contained 20 μl of DMSO.

FIG. 5 is a disk diffusion analysis for TSB007 (135B), showing theinhibition at 48 hours for Clostridium difficile NCTC 43593. The disk onthe left contained 20 μl of 10 mg/ml 135B in DMSO and the disk on theright contained 20 μl of DMSO.

FIG. 6 is a graph of the time-kill experiments of S. aureus NCTC 6571with TSB007 (NAL135B) at concentrations of 1.8 μg/ml and 3.6 μg/ml.Error bars represent standard deviations of triplicate experiments.

FIG. 7a-i shows tristyrylbenzene structural analogues of TSB007.

FIG. 8 shows distyrylbenzene structural analogues of TSB007.

FIG. 9 shows tetrastyrylbenzene structural analogues of TSB007.

FIG. 10 shows triacryloylbenzene structural analogues of TSB007.

FIG. 11 shows tricinnamylbenzene structural analogues of TSB007.

FIG. 12 shows tricinnamyltriazine structural analogues of TSB007.

EXAMPLES

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally equivalent products, compositions andmethods are clearly within the scope of the invention as describedherein.

Example 1 Synthesis of Compounds

The compounds of the present invention are synthesised using the HeckCross Coupling method.

Generally, the pathways for synthesis are as shown in FIGS. 1 and 2—thesynthesis of the target compounds through the Heck Cross couplingapproach. The reagents and reaction conditions for the chemicaltransformations shown in these figures are denoted by a letter for eachreaction. The key for this labelling is:

For FIG. 1: (a) Pd₂(dba)₃/[(t-Bu)₃PH]BF₄, Tetrahydrofuran (“THF”),reflux, 17 h (82-97%) (b) LiOH, H₂O/EtOH, reflux, 3 hrs (c) Pd/C—H₂,CH₂Cl₂/Et0H, 17 h (d) LiOH, H₂O/EtOH, reflux, 3 hrs

For FIG. 2: (a) NaOMe, MeOH, 22, rt. 17 h, 84% (b) Pd/C (10% W/W), H₂,1:1 CH₂Cl₂/MeOH, 17 h, rt, 93% (c) LiOH, MeOH/H₂O, reflux, 97% (d)Pd₂(dba)₃.CHCl₃, [(t-Bu)₃PH]BF₄, Cy₂NMe, THF, reflux, 17 h, 84% from 26,72 h, 29% from 27 (e) LiOH, 9:1 EtOH/H₂O, reflux, 17 h, 66% (f) Pd/C,H₂, 1:1 CH₂Cl₂/EtOH, 17 h, 96% (g) LiOH, EtOH/H₂O, reflux, 17 h, 82%.

General Conditions

Terephthaldehyde, tributylphosphonium tetrafluoroborate,1,3,5-tribromobenzene and 1,2,4-tribromobenzene were purchased from theSigma-Aldrich Chemical Company. Dry THF was distilled from sodiumbenzophenone ketyl radical and stored over a sodium mirror.N-Methyldicyclohexylamine was distilled under reduced pressure andstored under argon. Dess-Martin Periodinane, Herrmann-Beller paladacycle[Herrmann et al. Chem. Int. Ed. Engl. 1995, 34, 1844-1849] (methyl4-carboxybenzyl)triphenylphosphonium bromide, Pd₂(dba)₃.CHCl₃, andPd(PPh₃)₄ were prepared as described previously [Ukai et al. Organomet.Chem. 1974, 65, 253-266; Coulson, D. R. Inorg. Synth. 1972, 13,121-123]. 1,3-di(hydroxymethyl)benzene was prepared by the LiAlH₄reduction of dimethyl 1,3-benzenedicarboxylate in a similar procedure tothe 1,2-isomer [Sharpless, K. B.; Oi, R. Org. Synth. 1996, 73, 1-13]Ethyl 4-vinylbenzoate was prepared by the Fischer esterification of4-vinyl benzoic acid [Broos et al. J. Chem. Ed. 1978, 55, (12), 813. 33;Tullen et al. J. Chem. Ed. 1971, 48, (7)].

NMR spectra were acquired on either a Bruker AV500 (¹H at 500.13 MHz,¹³C at 125.8 MHz) or a Bruker AV600 (¹H at 600.13 MHz, ¹³C at 150.9 MHz)and all signals 8 are reported in parts per million (ppm). ¹H and ¹³Cassignments were made with the aid of DEPT, COSY, HSQC and HMBCsequences where appropriate. ¹H spectra were referenced to residual(partially) undeuterated solvents, CDCl₃ (CHCl₃ at 7.26 ppm) and d₆-DMSO(d₅-DMSO at 2.50 ppm (pentet)). ¹³C spectra were referenced to thedeuterated solvents, CDCl₃ at 77.16 ppm and d₆-DMSO at 39.52 ppm.

Infrared spectra samples were prepare using the KBr disc method andsamples acquired on a Perkin Elmer Spectrum One spectrometer at 2 cm⁻¹resolution. Electronic spectra were collected using a HP8452spectrophotometer in 1cm quartz cells at ˜1×10⁻⁵ or ˜1×10⁻⁶ MoIL⁻¹ inthe solvents indicated. Fluorescence spectra were recorded at 1×10⁻⁷MoIL⁻¹.

Mass spectra were acquired on a VG Autospec employing the electronimpact (EI) ionization mode.

Standard Conditions for Heck Cross-Coupling Procedure

To a flame-dried schlenk flask was added the halobenzene (1 equiv),Pd₂(dba)₃.CHCl₃ (2-15 mol %) and [(t-Bu)₃PH]BF₄ (10-60 mol %) which weresubsequently dried under vacuum for 15 minutes before being dissolved indry THF. N-Methyldicyclohexylamine (4 equiv) and ethyl 4-vinylbenzoate 7(3.3 equiv) were added via syringe and the reaction monitored by TLC(neat CH₂Cl₂). Upon completion of the reaction the residual THF wasremoved in vacuo, the crude material redissolved in CH₂Cl₂ and filteredto remove any insoluble material before being absorbed onto fine silicaand eluting with 0:100 to 2:98 MeOH/CH₂Cl₂.

Standard Method for Reduction of Alkenes

The alkene was loaded into a glass autoclave tube anddissolved/suspended in 1:1 CH₂Cl₂/MeOH or 1:1 CH₂Cl₂/EtOH depending uponthe ester present. Argon was bubbled through the mixture for 10 minutesbefore 10% Pd/C (˜10 wt % of alkene) was added, and the flaskpressurized with H₂ (50 atm). The reaction was allowed to proceed for 17h before being depressurized, purged with argon, filtered through a padof celite and concentrated under reduced pressure. Further purificationis described for each compound when necessary.

Standard Method for Saponification Reactions

The ester (1 equiv) and LiOH (2 equiv per ester) were dissolved in 9:1H₂O/MeOH or H₂O/EtOH depending upon the ester and refluxed overnight.After cooling to room temperature the solvent was removed under reducedpressure, the remaining solution diluted with H₂O, cooled in an ice-bathand the pH adjusted to 3 by the addition of HCl (1M). The precipitatewas collected filtered and product dried under vacuum.

1,3,5-Tris[(1E)-2′-(ethyl 4″-benzoate)vinyl]benzene (10)

Prepared as per the standard procedure using 1,3,5-tribromobenzene 8(1010 mg, 3.21 mmol), Pd₂(dba)₃.CHCl₃ (882 mg, 0.85 mmol), t-Bu₃PHBF₄(560 mg, 1.93 mmol), Cy₂NMe (3.0 mL), ethyl 4-vinylbenzoate 7 (1870 mg,10.61 mmol) and THF (40 mL). The product was eluted with 2:98MeOH/CH₂Cl₂ and recrystallized from CH₂Cl₂/EtOH to give 10 as an offwhite powder, 1.86 g (97%).

¹H NMR (600 MHz, CDCl₃): δ1.42 (t, J=7.1 Hz, 9H, CH₃), 4.40 (q, J=7.1Hz, 6H, CH₂), 7.24 (AB quartet, 6H, vinyl), 7.60 (d, J=8.3 Hz, 6H, ArH),7.61 (s, 3H, ArH), 8.06 (d, J=8.3 Hz, 6H, ArH).

¹³C NMR (150 MHz, CDCl₃): δ14.51 (CH₃), 61.13 (CH₂), 125.0 (CH), 126.5(CH), 128.7 (CH), 129.7 (C), 130.2 (CH), 130.5 (CH), 137.9 (C), 141.5(C), 166.5 (C═O).

IR (KBr): v (cm⁻¹) 2979, 2929, 1713, 1604, 1279, 1178, 1105, 762, 698

HR-EI⁺-MS: C₃₉H₃₆O₆ requires 600.2512 amu, found 600.2513.

EI⁺-MS: MI=C₃₉H₃₆O₆; m/z: 600.3 (100%)=MI⁺, 555.2 (7%)=[MI-EtO]⁺.

UV-Vis (CH₂Cl₂): λ (nm) [log ε(M⁻¹cm⁻¹)] 258 [4.49], 330 [4.28]

Fluorescence (CH₂Cl₂): excitation (nm) [emission (nm)] 258 [397, 418,518], 330 [397, 418]; (cyclohexane) 328 [393, 413]

1,3,5-Tris[(1E)-2′-(4″-benzoic acid)vinyl]benzene (5)

Using the standard saponification procedure, 10 (252.1 mg, 0.42 mmol),LiOH.H₂O (112.0 mg, 2.7 mmol) and 1:9 H₂O/EtOH (20 mL) gave angelatinous precipitate that was collected and recrystallised fromTHF/H₂O and dried to give the triacid 5 as a pale brown powder, 209 mg(95%).

¹H NMR (500.1 MHz, d₆-DMSO): δ7.49 (m, 6H, vinyl CH), 7.76 (d, J=8.5 Hz,6H, ArH), 7.88 (s, 3H, core ArH), 7.98 (d, J=8.5 Hz, 6H, ArH).

¹³C NMR (125.8 MHz, d₆-DMSO): δ (ppm) 125.0, 126.5, 128.4, 129.7, 129.9,130.50, 137.6, 141.3, 167.1.

IR (KBr): v (cm⁻¹) 3067, 3026, 1684 (v_(C═O)), 1604, 1566, 1420, 1384,1312, 1286, 1179.

HR-EI⁺-MS: C₃₃H₂₄O₆ requires 516.1573 amu, found 516.1564.

EI⁺-MS: MI=C₃₃H₂₄O₆; m/z: 516.1 (100%)=MI⁺, 472.1 (11.3%)=[MI-CO₂]⁺.

1,3,5-Tris[(1E)-2′-(ethyl 4″-benzoate)ethyl]benzene (11)

Conducted as per the standard reduction procedure with trimester 10 (251mg, 0.42), Pd/C (20 mg) and 1:1 CH₂Cl₂/EtOH (15 mL). The crude productwas recrystallized from CH₂Cl₂/EtOH to give the triester 11 237 mg (93%)of a white solid.

¹H-NMR (600 MHz, CDCl₃): δ1.38 (t, J=7.1 Hz, 9H, CH₃), 2.87 (m, 12H,bridge CH₂), 4.36 (q, J=7.1 Hz, 6H, CH₂), 6.76 (s, 3H), 7.20 (d, J=8.1Hz, 6H, ArH), 7.97 (d, J=8.1 Hz, 6H, ArH).

¹³C NMR (150 MHz, CDCl₃): δ14.4, 37.5, 38.0, 60.8, 126.5, 128.3, 128.6,129.7, 141.4, 147.2, 166.7.

HR-EI-MS: C₃₉H₄₂O₆ requires 606.2981 amu, found 606.2994.

1,3,5-Tris[2′-(4″-benzoic acid)ethyl]benzene (12)

Using the standard procedure triester 11 (252.0 mg, 0.42 mmol), LiOH.H₂O(107.2 mg, 2.6 mmol) and 1:9 H₂O/EtOH (20 mL) gave triacid 3 202 mg(93%) as a white powder.

¹H-NMR (500 MHz, d₆-DMSO): δ2.82 (cm, 12H, CH₂), 6.83 (s, 3H, ArH), 7.28(d, J=8.2 Hz, 6H, ArH), 7.85 (d, J=8.2 Hz, 6H, ArH).

¹³C NMR (125 MHz, CDCl₃): δ36.7, 37.1, 126.2, 128.4, 126.2, 129.3,140.9, 146.9, 167.3.

IR (KBr): v (cm⁻¹) 3067, 2929, 1686, 1610, 1422, 1315, 1288, 1179.

HR-EI⁺-MS: C₃₃H₃₀O₆ requires 522.2042 amu, found 522.5897.

EI⁺-MS: MI⁺=C₃₃H₃₀O₆; m/z: 504.2 (90%)=[MI-H₂O], 387.1(100%)=[MI-CH₂(C₆H₄CO₂H)].

1,2,4-Tris[(1E)-2′-(ethyl 4″-benzoate)vinyl]benzene (14)

Using the standard Heck cross-coupling procedure, 1,2,4-tribromobenzene13 (1.018 g, 3.2 mmol), ethyl 4-vinylbenzoate 9 (1.843 g, 10.5 mmol),Pd₂(dba)₃.CHCl₃ (87.6 mg, 0.08 mmol), [(t-Bu)₃PH]BF₄ (122.6 mg, 0.42mmol), Cy₂NMe (3 mL) in THF (40 mL) were heated for 17 h. The crudemixture was subjected to flash chromatography eluting with neat CH₂Cl₂.The crude product was recrystallised from CH₂Cl₂/EtOH to give 1.681 g(88%) of a pale yellow solid.

¹H NMR (600 MHz, CDCl₃): δ1.409, 1.413, 1.416 (3×t, J=7.1 Hz, 9H, CH₃),4.392, 4.394, 4.400 (3×q, J=7.1 Hz, 6H, CH₂) 7.06-7.11 (m, 2H, vinylCH), 7.17-7.28 (m, 2H, vinyl CH), 7.51-7.67 (cm, 9H), 7.73 (m, 1H, coreArH), 8.04- 8.09 (cm, 6H, ArH).

¹³C NMR (150 MHz, CDCl₃): δ14.5 (CH₃), 61.1, 61.13, 61.1 (CH₂), 125.8(CH), 126.5 (CH), 126.5 (CH), 126.6 (CH), 126.4 (CH), 127.3 (CH), 128.3(CH), 128.4 (CH), 128.7 (CH), 129.6 (C), 129.8 (C), 129.8 (C), 130.2(CH), 130.2 (CH), 130.3 (CH), 130.5 (C), 130.7 (C), 131.3 (C), 135.6(C), 136.4 (C), 136.9 (C), 141.62 (C═O), 141.66 (C═O), 141.7 (C═O).

IR (KBr): v (cm⁻¹) 2981, 1713 (V_(C═O)), 1604, 1278, 1178, 1107

HR-EI⁺-MS: C₃₉H₃₆O₆ requires 600.2512 amu, found 600.2504.

EI⁺-MS: MI=C₃₉H₃₆O₆; m/z: 600.2 (100%)=MI⁺, 555.2 (13.3%)=[MI-EtO]⁺,437.1 (70.1%)=[MI-2x OEt-EtO₂CH]⁺

UV-Vis (SoIv): λ (nm) [ε, (M⁻¹cm⁻¹)] 258 [4.53], 338 [4.83], 362 [4.76,shoulder].

Fluorescence (CH₂Cl₂): excitation (nm) [emission (nm)] 258 [436(shoulder), 450], 338 [436 (shoulder), 450]; (cyclohexane) 334 [416,440]

1,2,4-Tris[(1E)-2′-(4″-benzoic acid)vinyl]benzene (6)

Triester 14 (250 mg, 0.42 mmol) and LiOH.H₂O (70.2 mg, 1.67 mmol) in 9:1EtOH/H₂O were treated as described in the general saponifiactionprocedure giving the triacid 6 186.6 mg (86%) as a yellow/brown solid.

IR (KBr): v (cm⁻¹) 2929, 1684 (V_(C═O)), 1603, 1419, 1315, 1287, 1178,1125, 763.

1,2,4-Tris[2′-(ethyl 4″-benzoate)ethyl]benzene (15)

Triester 14 (306 mg, 0.51 mmol) and Pd/C (10% w/w, ca 40 mg) in 1:1EtOH/CH₂Cl₂ (20 mL) was treated as described. The crude product wasrecrystallised from THF and hexane to give ester 15 305 mg (98%).

¹H-NMR (600 MHz, CDCl₃): δ1.376, 1.381, 1.393 (3×t, 3×CH₃, 9H) 2.79-2.95(m, 12H), 4.33 -4.40 (m, 6H), 6.86 (s, 1H), 6.95 (d, J=7.8 Hz, 1H), 7.04(d, J=7.8 Hz, 1H), 7.17 (AB d, J=8.0 Hz, 2H), 7.19 (AB d, J=8.0 Hz, 2H)7.21 (AB d, J=8.1 Hz, 2H), 7.95 (cm, J=7.7 Hz, 6H)

¹³C NMR (150 MHz, CDCl₃): δ14.48, 14.50, 34.0, 34.4, 37.2, 37.7, 38.1,61.0, 61.0, 126.6, 128.43, 128.54, 128.56, 128.7, 129.5, 129.7, 129.8,129.9, 136.7, 139.0, 139.3, 147.2, 147. 2, 147.3, 166.7, 166.8, 166.8.

IR (KBr): v (cm⁻¹) 2981, 2942, 1713, 1610, 1283, 1177, 1123, 1108.

HR-EI⁺-MS: C₃₉H₄₂O₆ requires 606.2981 amu, found EI⁺-MS: MI=C₃₉H₄₂O₆;m/z: (%)=MI⁺, (%)=[MI-

]⁺

1,2,4-Tris[2′-(4″-benzoic acid)ethyl]benzene (16)

Triester 15 (200 mg, 0.33 mmol) and LiOH.H₂O (92.2 mg, 2.15 mmol) in 9:1EtOH/H₂O (25 mL) was treated as described in the general saponificationprocedure, giving triacid 16 161 mg (94%).

¹H NMR (500.1 MHz, d₆-DMSO): δ2.77-2.91 (m, 12H, methylene), 6.960 (AB,J=8.5 Hz, 1H), 6.967 (s, 1H), 7.07 (AB, J=8.5 Hz, 1H), 7.271 (AB, J=8.4Hz, 2H), 7.296 (AB, J=8.4 Hz, 2H), 7.299 (AB, J=8.4 Hz, 2H), 7.82-7.87(m, 6H), 12.83 (br s, CO₂H).

¹³C NMR (125.8 MHz, d₆-DMSO): δ33.1, 33.5, 36.3, 36.7, 37.0, 126.2,128.5, 128.6, 128.6, 128.6, 129.0, 129.3, 129.4, 136.4, 138.6, 138.7,146.9, 146.9, 167.3, 167.3.

IR (KBr): v (cm⁻¹), 1688, 1610, 1422, 1315, 1289, 1178.

HR-EI⁺-MS: C₃₃H₃₀O₆ requires 522.2042 amu, found 522.2045.

EI⁺-MS: MI=C₃₃H₃₀O₆; m/z: 522.2 (9%)=MI⁺, 504.2 (86%)=[MI-H₂O]⁺, 387.1(100%)=[MI-CH₂(C₆H₄CO₂H)]⁺.

1,2,4,5-Tetrakis[(1E)-2′-(ethyl 4″-benzoate)vinyl]benzene (18)

1,2,4,5-tetrabromobenzene 17 (255.8 mg, 0.66 mmol),ethyl-4-vinylbenzoate 9 (498.6 mg, 2.83 mmol), Pd₂(dba)₃.CHCl₃ (69.2 mg,0.07 mmol), [(t-Bu)₃PH]BF₄ (75.4 mg, 0.26 mmol), Cy2NMe (0.8 mL) in THF(8 mL) were treated under the aforementioned cross-coupling procedure.The product was purified by flash chromatography with CH₂Cl₂/MeOH(100:0-99:1) an the eluent. The crude material was recrystallized fromCH₂Cl₂/EtOH to give triester 18 541.2 mg (82%) as a bright yellow solid.

¹H NMR (600 MHz, CDCl₃): δ1.41 (t, J=7.1 Hz, 12H, CH₃), 4.40 (q, J=7.1Hz, 8H, CH₂), 7.13 (d, J=16 Hz, 4H, vinyl CH), 7.56 (d, J=16.1 Hz, 4H,vinyl CH), 7.60 (d, J=8.3 Hz, 8H, ArH), 7.83 (s, 2H, core ArH), 8.06 (d,J=8.3 Hz, 4H, ArH).

¹³C NMR (150 MHz, CDCl₃): δ14.3 (CH₃), 61.0 (CH₂), 125.2 (ArCH, core),126.5 (ArCH), 128.1 (CH, vinyl), 129.7 (C_(q)), 130.1 (ArCH), 131.0 (CH,vinyl), 135.6 (C), 141.4 (C), 166.3 (C═O).

IR (KBr): v (cm⁻¹) 2981, 1710, 1637, 1617, 1604, 1282, 1180, 1107.

HR-EI⁺-MS: C₅₀H₄₆O₈ required 774.3193 amu, found 774.3506 amu.

EI⁺-MS: MI=C₅₀H₄₆O₈; m/z: 774.4 (100%)=MI⁺, 729.1 (16%)=[MI-OEt]⁺, 611.1(45%)=[MI-CH₂(C₆H₄CO₂Et)]⁺.

UV-Vis (Solv): λ (nm) [log ε(M⁻¹cm⁻¹)] 258 [4.62], 312 [4.73], 346[4.82].

Fluorescence (CH₂Cl₂): excitation (nm) [emission (nm)] 258 [455, 518],312 [362, 381, 452], 346 [381, 454]; (cyclohexane) 336 [442].

1,2,4,5-Tetrakis[(1E)-2′-(4″-benzoic acid)vinyl)]benzene (7)

Triester 18 (151.7 mg, 0.20 mmol), LiOH.H₂O (86.6 mg, 2.06 mmol) in 9:1EtOH/H₂O (30 mL) were treated as described in the general saponificationprocedure. The crude product was recrystallized from THF and MeOH togive triacid 7 111.3 mg (85%) as a tan powder.

¹H NMR (500 MHz, CDCl₃): δ (d, J=Hz, 6H, ArH), (s, 3H, ArH), (d, J=Hz,6H, ArH)

¹³C NMR (125.8 MHz, CDCl₃): δ (ppm), 124.2, 127.1, 127.6 (CH), 129.5,129.9, 130.7, 135.3, 141.5, 167.2.

IR (KBr): v (cm⁻¹)

HR-EI⁺-MS: C₄₂H₃₀O₈ requires 662.1941 amu,

1,2,4,5-Tetrakis[(2′-(ethyl 4″-benzoate)ethyl)benzene (19)

Triester 18 (200.4 mg, 0.24 mmol) and Pd/C (10% w/w, c.a. 30 mg) in 1:1EtOH/CH₂Cl₂ (40 mL) was treated as described. The crude product wasrecrystallized from CH₂Cl₂ and EtOH to give compound 19 147.1 mg (79%)as a white solid.

¹H NMR (600 MHz, CDCl₃): δ1.38 (t, J=7.2 Hz, 12H), 2.82 (m, 16H), 4.36(q, J =7.2 Hz, 8H), 6.81 (s, 2H), 7.18 (d, J=8.0 Hz, 8H, ArH), (s, 3H,ArH), (d, J=Hz, 6H, ArH).

¹³C NMR (150 MHz, CDCl₃): δ14.8, 34.0, 37.8, 61.0, 128.5, 128.6, 129.8,130.6, 136.9, 147.2, 166.7.

IR (KBr): v (cm⁻¹) 2980, 2935, 1716, 1611, 1285, 1176, 1107, 1022.

HR-EI⁺-MS: C₅₀H₅₄O₈ requires 782.3819 amu,

1,2,4,5-Tetrakis[2′-(4″-benzoic acid)ethyl]benzene (20)

Triester 19 (101.4 mg, 0.13 mmol), LiOH.H₂O (51 mg, 1.22 mmol) in 9:1EtOH/H₂O (25 mL) were treated as described in the general saponificationprocedure, to give tetraacid 20 87.3 mg (97%) as a white solid.

¹H NMR (500.1 MHz, d₆-DMSO): δ2.77 (s, 16H, CH₂CH₂), 6.86 (s, 2H, H2),7.26 (AB, J=8.3 Hz, 8H, H2′) 7.84 (AB, J=8.3 Hz, 8H, H3′).

¹³C NMR (125.8 MHz, d₆-DMSO): δ33.1, 36.8, 128.5, 128.6, 129.4, 136.3,147.0, 167.3.

IR (KBr): v (cm⁻¹) 2945, 2863, 1688, 1610, 1422, 1315, 1288, 1178.

HR-EI⁺-MS: C₄₂H₃₈O₈ requires 670.2567 amu, found 670.2562.

EI⁺-MS: MI=C₄₂H₃₈O₈; m/z: 517.0 (100%)=[MI-H₂O—(CH₂(C₆H₄CO₂H]⁺, 499.0(12%)=[MI-2(H₂O)—(CH₂(C₆H₄CO₂H)]⁺, 381.0(19.5%)=[MI-H₂O-2(CH₂(C₆H₄CO₂H)]⁺, 135.0=[(CH₂(C₆H₄CO₂H)]⁺.

1,4-Bis[(1E)-2′-(ethyl 4″-benzoate)vinyl]benzene (28)

Method A: 1,4-Dibromobenzene 26 (101.5 mg, 0.43 mmol), ethyl 4-vinylbenzoate 7 (166 mg, 0.94 mmol), Pd₂(dba)₃.CHCl₃ (45.1 mg, 0.04 mmol),[(t-Bu)₃PH]BF ₄ (52.2 mg, 0.18 mmol), Cy₂NMe (300□l) in THF (5 mL) wereheated at reflux overnight. The THF was removed under reduced pressure,the crude product purified using flash chromatography with CH₂Cl₂ as theeluent. Additional recrystallisation from CH₂Cl₂ and EtOH, gave 28 152.2mg (84%).

Method B: 1,4-dichlorobenzene 27 (59.9 mg, 0.41 mmol), ethyl 4-vinylbenzoate 7 (160 mg, 0.91 mmol), Pd₂(dba)₃.CHCl₃ (42.1 mg, 0.04 mmol),[(t-Bu)₃PH]BF₄ (47.7 mg, 0.16 mmol), Cy₂NMe (300□l) in THF (5 mL) wereheated at reflux for 3 days. The reaction mixture was concentated underreduced pressure, the crude product was purified by chromatography withCH₂Cl₂ as the eluent. Additional recrystallisation from CH₂Cl₂ and EtOH,gave 28 49.8 mg (29%).

¹H NMR (600 MHz, CDCl₃): δ1.41 (t, J=7.1 Hz, 6H), 4.39 (q, J=7.1 Hz,4H), 7.14 (AB, J=16.3 Hz, 2H, vinyl CH), 7.24 (AB, J=16.3 Hz, 2H, vinylCH), 7.55 (s, 4H), 7.57 (d, J=8.1 Hz, 4H), 8.04 (d, J=8.1 Hz, 4H).

¹³C NMR (150 MHz, CDCl₃): δ14.5 (CH₃), 61.1 (CH₂), 126.5 (CH), 127.4(CH), 128.0 (CH), 129.5 (C), 130.2 (CH), 130.7 (CH), 136.9 (C), 141.8(C), 166.5 (C═O).

IR (KBr): v (cm⁻¹) 2984, 2925, 1716 (C═O), 1708 (C═O), 1279, 1179, 1107.

HR-EI⁺-MS: C₂₈H₂₆O₄ requires 426.1831 amu, found 426.1824 UV-Vis (Solv):λ (nm) [log ε(M⁻¹cm⁻¹)] 254 [3.85], 372 [4.74].

Fluorescence (CH₂Cl₂): excitation (nm) [emission (nm)] 254 [414, 437],372 [414, 437]; (cyclohexane) 328 [393, 413].

1,4-Bis[(1E)-2′-(4″-benzoic acid)vinyl]benzene (3)

Using the standard procedure 28 (150.7 mg, 0.35 mmol), LiOH.H₂O (62.3mg, 1.48 mmol) and 1:9 H₂O/EtOH (20 mL) gave an impure dark brown/blackprecipitate. The crude product was recrystallized from H₂O and EtOH togive acid 3 as a dark brown powder 86.2 mg (66%).

HR-EI⁺-MS: C₂₄H₁₈O₄ requires 370.1205 amu, found 370.1205.

1,4-Bis[2′-(methyl 4″-benzoate)vinyl]benzene (23)

(Methyl 4-carboxybenzyl)triphenylphosphonium bromide (22) (4.43 g, 9.02mmol) was dissolved in MeOH (100 mL) and treated with NaOMe (45 mL0.222M in MeOH). The ensuing yellow solution was treated withterephthalaldehyde (512 mg, 3.82 mmol) in one portion and the resultantmixture was heated at reflux for 17 h. The resulting yellow precipitateformed was collected and washed with MeOH to give 23 1.27 g (84%). 40:60mixture of the E/E and E/Z products.

¹H NMR (500.1 MHz, CDCl₃): δ3.834 (s, CH₃, EE), 3.837 (s, CH₃, EZ), 3.85(s, CH₃, EZ), 6.691 (AB, J=12.3 Hz, vinyl CH, EE), 6.726 (AB, J=12.3 Hz,vinyl CH, EZ), 6.735 (AB, J=12.3 Hz, vinyl CH, EE), 6.774 (AB, J=12.3Hz, vinyl CH, EZ), 7.10 (s, core ArH, EE), 7.23 (AB, J=8.3Hz, ArH, EZ),7.326 (AB, J=16.4 Hz, vinyl CH, EZ), 7.345 (AB, J=8.4 Hz, ArH, EE),7.389 (AB, J=16.4 Hz, vinyl CH, EZ), 7.393 (AB, J=8.4 Hz, ArH, EZ), 7.54(AB, J=8.3 Hz, ArH, EZ), 7.72 (AB, J=8.5 Hz, ArH, EZ), 7.84 (AB, J=8.4Hz, ArH, EE), 7.87 (AB, J=8.4 Hz, ArH, EZ), 7.85 (AB, J=8.5 Hz, ArH,EZ).

¹³C NMR (125.8 MHz, CDCl₃): δ52.1 (CH₃), 52.1 (CH₃), 126.6 (CH), 126.9(CH), 127.6 (CH), 128.2 (C), 128.2 (C), 128.3 (C), 128.7 (CH), 128.8(CH), 129.1 (CH), 129.2 (CH), 129.3 (CH), 129.4 (CH), 129.6 (CH), 130.7(CH), 131.6 (CH), 135.6 (C), 136.0 (C), 136.1 (C), 141.8 (C), 141.8 (C),142.0 (C).

IR (KBr): v (cm⁻¹) 3011, 2959, 1716, 1606, 1436, 1276, 1182, 1109.

HR-EI-MS: C₂₆H₂₂O₄ requires 398.1518 amu, found 398.1515 amu.

1,4-Bis[2′-(methyl 4″-benzoate)ethyl]benzene (24)

Diester 23 (1.27 g, 3.19 mmol) and Pd/C (10% w/w c.a 100 mg) in 1:1MeOH/CH₂Cl₂ (30 mL) was treated as described in the general proceduresection. The crude product was recrystallized from CH₂Cl₂ and MeOH togive 1.20 g (93%) as a white solid.

¹H NMR (600 MHz, CDCl₃): δ2.93 (AA′BB′, 8H, CH₂CH₂), 3.91 (s, 6H, OCH₃),7.05 (s, 4H, H2/H3), 7.21 (d, J=7.6 Hz, 4H, H2′), 7.95 (d, J=7.6 Hz, 4H,H3′).

¹³C NMR (150 MHz, CDCl₃): δ (ppm) 37.2, 38.0, 52.1, 128.1, 128.6, 128.7,129.8, 139.0, 147.3, 167.3.

IR (KBr): v (cm⁻¹) 2944, 2923, 1726, 1609, 1431, 1280, 1176, 1105.

HR-EI⁺-MS: C₂₆H₂₆O₄ requires 402.1831 amu, found 402.1834 amu.

EI⁺-MS: MI=C₂₆H₂₆O₄; m/z: 402.2 (8%)=MI⁺, 370.1 (33.2%)=[MI-MeOH]⁺,253.1 (100%)=[MI-CH₂C₆H₄CO₂Me]⁺, 149.1=[CH₂C₆H₄CO₂Me]⁺.

1,4-Bis[2′-(4″-benzoic acid)ethyl]benzene (25)

Method 1: Diester 24 (308.6 mg, 0.77 mmol), LiOH.H₂O (125 mg, 3.0 mmol)in MeOH/H₂O (9:1, 20 mL) were treated as described in the generalsaponification procedure, to provide diacid 25 280 mg (97%).

Method 2: Diacid 3 (107.2 mg, 0.25 mmol) and Pd/C (10% w/w c.a. 10 mg)in 1:1 EtOH/CH₂Cl₂ (10 mL) was treated as described. The crude mixturecontaining 29 (103 mg, 96%) was suspended in 9:1 EtOH/H₂O (10 mL) andLiOH.H₂O (26.3 mg, 0.63 mmol) added and treated under the generalsaponification procedure described earlier to afford diacid 3 (73.2 mg,82%)

¹H NMR (500.1 MHz, d₆-DMSO): δ2.88 (AA′BB′, 8H, CH₂CH₂), 7.11 (s, 4H,H2/H3), 7.32 (d, J=8.2 Hz, 6H, ArH), 7.85 (d, J=8.2 Hz, 6H, ArH).

¹³C NMR (125.8 MHz, d₆-DMSO): 36.2, 37.0, 128.3, 128.6, 129.3, 138.7,146.8, 167.3.

IR (KBr): v (cm⁻¹) 2944, 2923, 1685 (C═O), 1610, 1425, 1318, 1292, 1180,537.

HR-EI⁺-MS: C₂₄H₂₂O4 requires 374.1518 amu.

1,3-Bis[2′-(methyl 4″-benzoate)vinyl]benzene (4)

Isophthaldehyde 30 (460 mg, 3.43 mmol), xx (4.4 g, 8.95 mmol), NaOMe (20mL, 1.0 M) in MeOH (100 mL) were treated under analogous conditions tothose described for the preparation of alkene 23. The white precipitatewas filtered, washed with MeOH, and dried to give 4 (0.85 g, 62%). Theproduct was a 44:56 mixture of the E/E and E/Z products.

¹H NMR (600.1 MHz, CDCl₃): δ3.82 (s, CH₃, ct), 3.83 (s, CH₃, E/E), 3.85(s, CH₃, E/Z), 6.64 (AB, J=12.4 Hz, trans vinyl CH, E/E), 6.67 (AB,J=12.4 Hz, trans vinyl CH, E/E), 6.76 (AB, J=12.3 Hz, trans vinyl CH,E/Z), 6.81 (AB, J=12.3 Hz, trans vinyl CH, E/Z), 7.05 (dd, J₁=7.6 Hz,J₂=1.4 Hz, H4, ee), 7.08 (s, H2, ee), 7.10 (d, J=7.7 Hz, H4/H6, E/Z)7.13 (AB, J=16.5 Hz, cis vinyl CH, E/Z), 7.17 (t, J=7.6 Hz, H5, E/E),7.25 (AB, J=8.2 Hz, H2′, E/Z), 7.27 (t, J=7.7 Hz, H5, E/Z), 7.33 (AB,J=16.5 Hz, cis vinyl CH, E/Z), 7.36 (AB, J=8.2 Hz, ArH, E/Z), 7.46 (s,H2, E/Z), 7.50 (d, J=7.7 Hz, H4/H6, E/Z), 7.66 (AB, J=8.3 Hz, ArH, E/Z),7.76 (AB, J=8.4 Hz, H3′, E/E), 7.84 (AB, J=8.3 Hz, ArH, E/Z), 7.92 (AB,J=8.4 Hz, ArH, E/Z).

¹³C NMR (150.9 MHz, CDCl₃): δ52.1 (CH₃), 52.1 (CH₃), 126.2 (CH), 126.6(CH), 127.0 (CH), 127.6 (CH), 127.9 (CH), 128.1 (C), 128.2 (C), 128.3(C), 128.3 (CH), 128.3 (CH), 128.7 (CH), 128.9 (CH), 128.9 (CH), 129.2(CH), 129.3 (CH), 129.3 (CH), 129.5 (CH), 129.6 (CH), 130.9 (CH), 131.7(CH), 131.8 (CH), 136.6 (C), 136.8 (C), 136.8 (C), 141.6 (C), 141.9 (C),165.9 (C), 165.9 (C), 166.0 (C).

IR (KBr): v (cm⁻¹) 1720, 1606, 1435, 1280, 1179, 1109.

HR-EI⁺-MS: C₂₆H₂₂O₄ requires 398.1518 amu, found 398.1518

1,3-Bis[2′-(methyl 4″-benzoate)ethyl]benzene (31)

The E/Z isomeric mixture 4 (499.3 mg, 1.25 mmol) and Pd/C (10% w/w, c.a.˜50 mg) in MeOH/CH₂Cl₂ (40 mL, 1:1) was treated as described previously.The crude product was recrystallized from CH₂Cl₂/MeOH to give ester 31(489.7 mg, 98%) as a white solid.

¹H-NMR (600 MHz, CDCl₃): δ2.8-2.96 (AA′BB′, 8H, CH₂CH₂), 3.90 (s, 6H,Me), 6.91 (s, 1H, H2), 6.99 (dd, 2H, J=7.6 and 1.4 Hz, 2H, H4/H6),7.16-7.25 (cm, 5H, ArH), 7.96 (d, J=8.2 Hz, 4H, H3′).

¹³C NMR (150 MHz, CDCl₃): δ37.5, 38.0, 52.1, 126.3, 128.0, 128.5, 128.7,128.8, 129.8, 141.3, 147.3, 167.2.

IR (KBr): v (cm⁻¹) 1715, 1607 (m), 1438, 1279, 1109.

HR-EI⁺-MS: C₂₆H₂₆O₄ requires 402.1831 amu, found 402.1840.

1,3-Bis[2′-(4″-benzoic acid)vinyl]benzene (32)

Ester 25 (202.6 mg, 0.50 mmol), LiOH.H₂O (92.2 mg, 2.20 mmol) and 9:1MeOH/H₂O (30 mL) were treated as described in the general saponficationprocedure, giving 175.8 mg (94%) as a white solid.

¹H-NMR (500.1 MHz, d₆-DMSO): δ2.87 (AA′BB′, 8H, CH₂CH₂), 6.99-7.05 (m,3H, ArH) 7.15 (t, J=7.4 Hz, 1H, H5), 7.31 (AB, J=8.4 Hz, 4H, H2′), 7.84(AB, J =8.4 Hz, 4H, H3′), 12.8 (br s, 2H, CO₂H).

¹³C NMR (125.8 MHz, d₆-DMSO): 36.5, 37.0, 126.0, 128.1, 128.4, 128.6,128.6, 129.3, 141.0, 146.9, 167.3.

HR-EI⁺-MS: C₂₄H₂₂O₄ requires 374.1518 amu, found 374.1523.

EI⁺-MS: MI=C₂₄H₂₂O₄, m/z: (%)=MI⁺, 239.0 (100%)=[MI-(CH₂(C₆H₄CO₂H)]⁺,193.0 (34%)=[MI-(CH₂(C₆H₄CO₂H)—CO₂H—H⁺]⁺.

Example 2 Toxicity of a Compound of Formula C—TSB007 (135B) (A)Haemolytic Activity

In a preliminary study using disk diffusion methodology, a compound ofFormula C (TSB007 also known as “135B”) (10 mg/ml) had antibacterialactivity mostly against Gram-positive bacteria.

As an indicator of toxicity to human cells, haemolysis experiments onsheep erythrocytes were undertaken with TSB007.

10 mg/ml stock solution of TSB007 was prepared in 100% DMSO. Masterstock solutions of TSB007 (10 mg/ml) were made by dissolving 20 mgdehydrated compound in 2 ml 100% DMSO. They were stored in foil-coveredglass bottles at −20° C. Master stocks stored in this way retained fullantimicrobial activity for a minimum period of 6 weeks (results notshown).

Serial 10-fold dilutions were performed in PBS to make solutions of1000, 100, 10 and 1 mg/L of TSB007. In microcentrifuge tubes, 500 μl ofeach dilution was combined with 480 μl PBS and 20 μl washed sheeperythrocytes (100%) so that the final concentration of erythrocytes was2% and the final concentrations of compound were 500, 50, 5 and 0.5mg/L. Dilutions of DMSO without TSB007 were prepared and tested as aboveto check for haemolysis due to DMSO. A 100% haemolysis control wasprepared with 980 μl water and 20 μl erythrocyte suspension. A negativecontrol was prepared with 980 μl PBS and 20 μl erythrocyte suspension.Dilutions and controls were prepared and tested in duplicate.

Tubes were incubated at 37° C. for 2 h on a rocker then centrifuged at12 000 g for 5 mins. The OD₅₄₀ of the supernatant was determined using100 μl volumes transferred to a microtitre tray. % haemolysis wasdetermined by blanking the OD against that of the negative control andpresenting the resulting OD as a proportion of the OD of the positivecontrol (blanked with water).

The haemolytic activity of TSB007 was very low (Table 1). No haemolysiswas observed for the equivalent tests of DMSO.

TABLE 1 Haemolysis of sheep erythrocytes exposed to 0.5-500 mg/L ofcompound TSB007 Concentration of Average (SD) % haemolysis compound(mg/L) (n = 2) 500 5.053 (0.595) 50 2.316 (0.099) 5 2.175 (0.099) 0.5−2.316 (0.695)

(B) Cytotoxic Activity

L929 cells grown to approximately 80% confluency in HGM-M were washedwith Hanks, trypsonised to detach the cells from the flask, then dilutedto 10⁵ cells/ml in HGM-M and 200 μl used to inoculate the wells of a96-well microtitre tray. After incubation for 24 h at 37° C., adherentcells were washed with Hanks and then 100 μl HGM-M was added to thewells. Fresh TSB007 master stock solution (10 mg/ml) was serially10-fold diluted in HGM-M to make solutions of 1000, 100, 10 and 1 μg/mlof TSB007. To the wells of the microtitre tray, 100 μl of these TSB007solutions were also added, giving final concentrations of 500, 50, 5 and0.5 μg/ml of TSB007. The same dilutions of DMSO were prepared and testedto check that cytotoxic activity was not due to the DMSO. A negativecontrol was prepared containing only HGM-M, and a positive control wasprepared by adding 100 μl of carboplatin (10 mg/ml; Mayne Pharma PtyLtd, Australia) to wells containing 100 μl HGM-M. Controls and dilutionsof TSB007 and DMSO were prepared in duplicate.

After 24 h shaking at 37° C., cytotoxicity was quantified using theneutral red assay. The cells were washed with Hanks then 200 μl HGM-Mand 20 μl Neutral Red (3.3 g/L; Sigma-Aldrich) were added to each well.After 2 h shaking at 37° C., cells were washed twice with PBS then 200μl of 1% acetic acid in 50% EtOH was added to each well to solubilisethe stain. After 15 min shaking at 37° C. the OD_(690 nm) of the wellswas measured and subtracted from the OD_(540 nm) Results were thenblanked against wells to which no cells had been added, and converted toa ratio of the OD of the negative control. Ratios ≦0.5 indicated acytostatic effect. Testing was performed on two separate occasions.

At concentrations of 0.5-50 μg/ml, TSB007 was not significantlycytostatic to L929 cells (Table 2). The compound was cytostatic at 500μg/ml. The concentrations of DMSO present in the different TSB007dilutions were not significantly cytostatic (average ratios ≧0.515). Thecontrols performed as expected.

TABLE 2 Cytotoxicity of 0.5-500 μg/ml TSB007 to L929 cells Concentrationof Average (SD) compound (μg/ml) (n = 2)^(a) 500 0.384 (0.009) 50 0.867(0.030) 5 0.882 (0.021) 0.5 0.846 (0.285) ^(a)The ratio of the well's ODcompared to the OD of the negative control. Ratios ≦ 0.5 indicate acytostatic effect and are highlighted.

(C) Ames Test for Mutagenic Activity

To perform the Ames Test on TSB007, a method based on that published byZeiger and Mortelmans in Current Protocols in Toxicology (1999; Section3.1.1-3.1.29) was used, in the absence of a metabolic activation system.

Briefly, single colonies from overnight BA cultures of three commonlyused Salmonella tester strains, S. typhimurium TA98, TA100 and TA1535,were used to inoculate 10 ml of nutrient broth. After incubation for15-18 h at 37° C. with shaking, the concentration of the culture wasappropriate for use in the test (˜1-2×10⁹ cfu/ml).

Glucose minimal agar plates with a volume of 20 ml were driedthoroughly. Molten top agars (2 ml) were prepared, supplemented withbiotin and trace histidine, and maintained at 43-48° C. To these, 50 μlof the bacterial broth culture (˜1×10⁸ cells) and 100 μl of testsolution (see below) were added. The molten top agar was then poureddirectly over the surface of the glucose minimal agar and gently swirledto ensure even distribution of the agar. Once solidified, plates wereincubated at 37° C. for 48 h then colony counts were performed. Plateswere prepared in duplicate.

Test solutions were prepared as follows and included threeconcentrations of TSB007, a negative solvent control and a positivecontrol (selected from the recommended positive control chemicals andtest concentrations) for each strain. Master stock solution (10 mg/ml)was incorporated directly into molten top agar to test the compound at1000 μg/plate. Dilutions of the stock were prepared in sterile distilledwater to also test 100 μg/plate and 300 μg/plate. DMSO (100%) wasincorporated into molten top agar as the negative solvent control. Thiswas equivalent to the highest amount of DMSO incorporated into themolten top agar when testing TSB007. The positive controls used were4-nitro-o-phenlenediamine at 2.5 μg/plate (for TA98), and sodium azideat 5 μg/plate (for TA100 and TA1535).

Colony counts that were two to three times greater than on the negativesolvent control plate indicated a mutagenic effect and were regarded as‘positive’. In these cases, the increase in colonies is usually doserelated. A ‘positive’ result in this test is highly predictive of rodentcarcinogenicity.

TSB007 gave a ‘negative’ result (Table 3). In general, the number ofcolonies on plates containing TSB007 was not greater than on thenegative control plates. The positive control performed as expected. Thedecrease in colony counts at 1000 μg/plate TSB007 may be due toantimicrobial activity against the tester strains.

TABLE 3 Colony counts of three S. typhimurium tester strains exposed todifferent concentrations of TSB007 under Ames Test conditions Number ofcolonies Nega- Posi- tive tive TSB007 100 TSB007 300 TSB007 1000 StrainControl Control μg/plate μg/plate μg/plate TA98 14 300 13 11 2 12 285 1611 3 TA100 113 350 111 110 88 96 360 92 105 89 TA1535 28 600 17 16 0 29600 29 24 0

Example 3 Antimicrobial activity of Formula C (A) Initial Screening byDisk Diffusion

Studies using disk diffusion methodology showed that the compound ofFormula C, (“TSB007”) (10 mg/ml) had antibacterial activity against arange of organisms, mostly Gram-positive bacteria.

Briefly, blood agar plate (BA) cultures of the 38 organisms listed inTable 5 were prepared over 24 and 48 h for normal and slow-growingorganisms, respectively. Pre-dried (30 mins) Mueller Hinton agar plates(MHA; from PathWest Media) were swabbed with 0.5 McFarland suspensionsof the BA cultures in saline (0.85% NaCl) as per CLSI guidelines.Anaerobes were instead inoculated onto pre-reduced BA plates, andStreptococcus spp. onto MHA containing 5% sheep blood. Two Whatman 6 mmAntibiotic Assay disks were placed onto each inoculated plate. One diskwas impregnated with 20 μl 100% DMSO and the second with 20 μl of 10mg/ml TSB007 in 100% DMSO. Plates were incubated at 35° C. (with 5% CO₂where mentioned in the Table). Zones were measured after 24 and 48 h.

TSB007 resulted in zones of inhibition of all Gram-positive bacteriatested (Table 5). Zones halved with S. aureus strains, and were nolonger seen with S. xylosus, following extended incubation (24 h vs. 48h), mostly due to the growth of subpopulations of discrete colonies. Nozones of inhibition were seen with C. albicans nor with Gram-negativebacteria except M. catarrhalis. Y. enterocolitica had a negligible zoneof inhibition after 24 h that was not seen after 48 h.

Photographs were taken after 48 h of all plates where TSB007 producedzones of inhibition. FIGS. 3-5 show a selection of these.

TABLE 5a Disk diffusion zone diameters for compound TSB007 (10 mg/ml, 20μl) and a DMSO control tested against a panel of Gram-negative bacteria

Note. Grey shading indicates no activity. No result indicates theorganism was not tested.

TABLE 5b Disk diffusion zone diameters for compound TSB007 (10 mg/ml, 20μl) and a DMSO control tested against a panel of Gram-positive bacteriaand Candida albicans

Note. Grey shading indicates no activity. No result indicates theorganism was not tested.

(B) Anaerobic Screening

While the above study showed compound TSB007 to be active againstGram-positive bacteria, the zones of inhibition were smaller for theanaerobic Gram-positives. The Gram-negative anaerobe was not inhibitedby TSB007. The compound may therefore be less active in an anaerobicenvironment. Staphylococcus aureus is a facultative anaerobe. Themembrane potential is reduced for this organism when grown anaerobicallyand therefore can affect susceptibility to certain antibiotics.

To confirm the compound was also active against S. aureus in ananaerobic environment, zones of inhibition of S. aureus grownaerobically and anaerobically were compared.

The method described above was used for this investigation except thattwo MHA plates were prepared for each organism with one examined afteraerobic incubation and the other after anaerobic incubation. Theorganisms tested included S. aureus NCTC 6571, S. aureus NCTC 10442, andS. epidermidis ATCC 12228. The results are shown in Table 6. Zones ofinhibition were larger when these organisms were incubated anaerobicallycompared to aerobically. This was largely due to a lack, when testedanaerobically, of the “creeping” zones (a zone of faint growth withinthe main zone of inhibition) seen on plates incubated aerobically. Nozones of inhibition were seen for DMSO only.

TABLE 6 Disk diffusion zone diameters for compound TSB007 (10 mg/ml, 20μl) against Staphylococcus spp. incubated aerobically and anaerobicallyZone diameters (mm) 24 h 48 h Organism O₂ AnO₂ O₂ AnO₂ S. aureus NCTC6571 14 18 No zone 18 S. aureus NCTC 10442 14 17 11 17 S. epidermidisATCC 12228 18 22 18 22

Example 4 MIC and MBC by Broth Microdilution (A) MIC and MBC by BrothMicrodilution

The method used was based on CLSI protocols for broth microdilutiontesting of these species. As recommended by CLSI, inocula were preparedby direct colony suspension, and the media used were cation-adjustedMueller Hinton broth (CA-MHB) for Staphylococcus spp. and CA-MHBcontaining 5% lysed horse blood (CA-MHB+LHB) for Streptococcus spp.Briefly, 24 h blood agar plate (BA) cultures of Staphylococcus aureusstrains NCTC 6571, ATCC 29213, ATCC 33592 (MRSA) and NCTC 10442 (MRSA),Staphylococcus epidermidis ATCC 29971, Staphylococcus xylosus ATCC29971, Streptococcus pneumoniae strains ATCC 49619 and ATCC 6305, andStreptococcus pyogenes strains NCTC 8191 and NCTC 8302 were used toprepare 0.5 McFarland suspensions in saline. Two 640 μg/ml workingstocks of TSB007 were prepared from a fresh master stock, one in CA-MHBfor testing against the Staphylococcus spp. and the other in CA-MHB+LHBfor testing against Streptococcus spp. The working stocks were furtherdiluted 1/10 in these broths for adding to microtitre trays to test MICsof 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.06 μg/ml.

The wells of two 96-well microtitre trays (one for Staphylococcus spp.and one for Streptococcus spp.) were filled with 100 μl of theappropriate broth, except for the first column. The last column, asterility control containing no TSB007 or inoculum, was filled with anadditional 100 μl broth. Then 100 μl of the 64 μg/ml TSB007 solution wasadded to the first two columns, the contents of column 2 was mixed, then100 μl of this was used in two-fold serial dilutions along the rest ofthe tray except the last two columns. The final 100 μl was discarded.The second last column was used as a growth control so did not haveTSB007 added. Finally, to the first 11 wells of one row per strain, 100μl of a 10⁻² dilution (in the appropriate broth) of the 0.5 McFarlandsuspension was added. The microtitre trays were then incubated at 35° C.for 24 h before the MICs were read visually. Wells were furthersubcultured (10 μl drop onto BA then incubated at 35° C. for 24 h) andcolonies subsequently counted to determine minimum bactericidalconcentrations (MBCs). The concentration of the initial inoculum wasconfirmed on BA by counting colonies from 2×10 μl spots of 10⁻⁵dilutions of the 0.5 McFarland suspensions. Testing was performed on twoseparate occasions.

The concentrations of inocula were largely as expected, equating to1.0×10⁵-6.0×10⁵ CFU/ml. Slightly lower concentrations were achieved forS. xylosus ATCC 29971 (5.0-7.5×10⁴ CFU/ml) and S. pneumoniae ATCC 49619(2.5-5.0×10⁴ CFU/ml). TSB007 was more active against Staphylococcus spp.(particularly S. aureus) than Streptococcus spp. This was consistentwith disc diffusion results above. S. aureus and S. epidermidis had thelowest MICs (4 μg/ml). In general, MBCs were significantly higher thanMICs.

TABLE 4 MIC and MBC of compound TSB007 tested against Staphylococcusspp. and Streptococcus spp.^(a) Final inoculum MIC MBC Organism (×10⁵CFU/ml) (μg/ml) (μg/ml) S. aureus NCTC 6571 3.25-3.75 2-4 >32 S. aureusATCC 29213 4.50-5.00 4 >32 S. aureus ATCC 33592 (MRSA) 4.75-6.00 2-4 >32S. aureus NCTC 10442 (MRSA) 2.25-3.25 2-4 >32 S. epidermidis ATCC 122282.00-3.25 2-4 >32 S. xylosus ATCC 29971 0.50-0.75  8-16 8-16 S.pneumoniae ATCC 49619 0.25-0.50 32 >32 S. pneumoniae ATCC 63051.00-2.00 >32 >32 S. pyogenes NCTC 8191 1.00-2.50 >32 >32 S. pyogenesNCTC 8302 1.00-1.50 >32 >32 ^(a)Results are from two independentreplicates

(B) Expansion of Broth Microdilution MIC and MBC Testing Range for S.aureus NCTC 6571 and E. coli NCTC 10418

The method described above, with some modifications, was used todetermine the MIC and MBC for S. aureus NCTC 6571 and E. coli NCTC10418. Concentrations of the compound were previously too low todetermine the MBC of this S. aureus strain.

Modifications were the use of Mueller-Hinton broth (MHB) instead ofCA-MHB, the preparation of bacterial inoculum by the growth methodinstead of direct colony suspension, and the use of more concentratedworking stocks and test concentrations of TSB007. Hence, overnight 10 mlMHB cultures prepared from BA cultures were used to make the 0.5McFarland suspensions in MHB, and doubling dilutions of the workingstock at 2048 μg/ml TSB007 were made in MHB across the tray to test MICsof 1024 μg/ml to 0.5 μg/ml. Testing was performed on two separateoccasions.

Subsequently, an extended range of concentrations of TSB007 was testedfor inhibition of S. aureus NCTC 6571 and E. coli NCTC 10418. The MIC ofS. aureus NCTC 6571 was 2 μg/ml, confirming the previous MIC result forthis strain, and the MBC was 128-256 μg/ml. The MIC and MBC for E. coliNCTC 10418 were 512 μg/ml and >1024 μg/ml, respectively.

Example 5 Time-kill Experiments

An overnight 10 ml MHB culture of S. aureus NCTC 6571 was diluted to 0.5McFarland in MHB then 1 ml was used to inoculate each of four 100 mlflasks prepared as follows:

-   -   1) 9.0 ml MHB (control)    -   2) 9.99 ml MHB plus 9.7 μl of TSB007 working stock at 2048 μg/ml        (1.8 μg/ml )    -   3) 9.98 ml MHB plus 19.5 μl of TSB007 working stock at 2048        μg/ml (3.6 μg/ml )    -   4) 6.74 ml MHB plus 256 μl of a TSB007 master stock (320 μg/ml        ). This equated to 3.2% DMSO which did not have any effect on        the viability of S. aureus NCTC 6571 (data not shown).

The flasks were incubated at 37° C. for 10 min before inoculation.Samples (0.1 ml) were taken immediately after inoculation (time 0) andthen at 15, 30, 60, 120 and 240 min. The samples were immediatelydiluted 10⁻¹ in 0.01 M phosphate buffered saline at pH 7.0 (PBS).Further 10-fold dilutions in PBS were made down to 10⁻⁴ until the 60 mintime point, and then down to 10⁻⁶ for the remaining time points. Viablecounts were estimated using 3×20 μl drops from each dilution on MHAwhich was then incubated at 37° C. overnight before counting. These werenormalised against the initial counts from each flask. Testing wasperformed on three separated occasions.

The viability of S. aureus NCTC 6571 cultures containing TSB007 at 1.8μg/ml (NB. The MIC was 2 μg/ml ), 3.6 μg/ml (1.8×MIC) and at 320 μg/ml(1.25×MBC) was tested. The initial concentration of microorganism ineach flask was 0.5-1.0×10⁷ CFU/ml. At both 1.8 μg/ml and 3.6 μg/ml,TSB007 was inhibitory for the 4 h of the experiment (FIG. 6). At 24 h,the cells with TSB007 at 1.8 μg/ml had grown to counts of 10⁸ CFU/ml,and those with 3.6 μg/ml to 10³-10⁶ CFU/ml. The cells with 320 μg/ml atthe first time point of 15 min were below the limit of detection witha >99.9% kill.

Example 6 Analogues of TSB007

Various analogues of TSB007 were synthesised and tested in DMSO in diskdiffusion assays against S. aureus NCTC 6571 using the protocol ofExample 3. A number of compounds were also tested against E. coli NCTC10418.

TABLE 5 Compounds tested (see FIGS. 8 to 12 for structure information)Zone of inhibition (mm) Compound S. aureus NCTC 6571 E. coli NCTC 10418TSB049 0 0 TSB063 0 0 TSB037 0 0 TSB041 0 0 TSB019 0 0 TSB023 0 0 TSB0250 0 TSB065 14 0 TCT003 0 0 TSB001 0 — TSB009 0 — TSB053 0 — TAB001 0 —TCB001 11 — TCB003 0 — 0 — — Not tested

Compounds TSB065 and TCB001 were successful at inhibiting growth of S.aureus NCTC 6571, with TSB065 giving a zone of inhibition of 14 mm andTCB001 giving a zone of inhibition of 11 mm.

Several analogues were also tested against a range of microorganisms asshown in Table 6.

TABLE 6 Effect of some analogues of TSB007 on growth of variousmicrooganisms. Zone of Inhibition (mm) of compounds Microorganism TSB068TSB067 QSB001 Gram −ve bacteria A. calcoaceticus ATCC 15308 0 0 0 A.hydrophila NCTC 8049 7 7 7 C. freundii NCTC 9750 0 0 8.5 E. coli NCTC10418 0 6.5 7 E. coli NCTC 10538 0 7.5 7.5 K. pneumoniae NCTC 10896 0 08 M. catarrhalis NCTC 3625 32 32 18 P. mirabilis NCTC 10975 0 0 0 P.aeruginosa NCTC 10662 0 0 0 S. typhimurium ATCC 13311 0 0 8.5 S.flexneri NCTC 8192 0 0 0 S. maltophilia ATCC 13637 0 0 8 V. choleraenon-01 clinical 0 0 8 Y. enterocolitica clinical 26609 0 0 7.5 Gram +vebacteria B. cereus ATCC 13061 0 0 0 B. subtilis ATCC 6633 0 0 0 E.faecalis NCTC 775 0 0 0 L. monocytogenes NCTC 7973 15 12 0 M. luteusATCC 10240 8 20 0 S. aureus NCTC 6571 8 14 8.5 S. aureus NCTC 10442 0 120 S. epidermidis ATCC 1228 0 11 0 S. xylosus ATCC 29971 0 0 0 S.pyogenes NCTC 8191 20 14 8 S. pneumoniae ATCC 49619 23 16 9 Fungi C.albicans ATCC 90028 0 0 0

Componds DSB001, DS 002, DSB004, TSB008 and QSB002 showed no zones ofinhibition with S aureus NCTC6571 when applied at 10 mg/ml.

1.-16. (canceled)
 17. A compound of Formula (I):

wherein R^(B) and R^(C) are independently C₂-alkyl, C₂-alkenyl, orC₂-alkynyl; R^(A) and R^(D) are H or COOH or an ester thereof, and atleast one of R^(A) and R^(D) is COOH or an ester thereof.
 18. Thecompound of claim 17, wherein R^(A) and R^(D) are independently COOCH₃or COOCH₂CH₃.
 19. The compound of claim 17, wherein R^(B) and ^(RC) arethe same.
 20. The compound of claim 19, wherein R^(A) and R^(D) are thesame.
 21. The compound of claim 17, wherein R^(A) and R^(D) are thesame.
 22. The compound of claim 17, wherein R^(B) and R^(C) are eachC₂-alkenyl.
 23. The compound of claim 22, wherein R^(B) and ^(RC) aretrans C₂-alkenyl.